Autoimmune mechanisms have long been implicated in the pathogenesis of human dilated cardiomyopathy (DCM). The recent development of sensitive molecular and biochemical techniques has provided some clues to the etiology and identification of potential autoantigen(s); thus, detection of coxsackievirus B3 RNA transcripts by in situ hybridization and PCR techniques in cardiac tissues and myocytes of patients post myocarditis and DCM, coupled with the finding that the sera from DCM patients contain high titers of antibodies against the adenine nucleotide translocator, ANT (a mitochondrial protein), for the first time provide a basis for the study of the pathogenic mechanisms of human DCM. The significance of such anti-ANT antibodies is highlighted not only by the finding that rabbit anti-ANT sera induce cardiac myocyte damage in vitro but also by the observation that experimental immunization of guinea pigs with ANT leads to severe cardiac dysfunction. Our laboratory confirms the finding that sera from select cases of DCM do react with the native ANT. In addition, however, these sera in our lab have been shown to react with the branched chain alphaketoacid dehydrogenase (BCKD) complex of mitochondrial proteins and with cardiac myosin. Immunohistological studies by our lab have shown that affinity-purified rabbit and human DCM sera specific for ANT and BCKD appear to intensely stain small focal patchy areas of the myocardium only in tissue specimens from DCM patients. Our lab has also cloned, sequenced, and prepared full-length recombinant proteins of the human ANT and BCKD (and fragments thereof). A series of cloned human T cell lines isolated from DCM cardiac tissues which specifically react with ANT, BCKD, and myosin has also been prepared. While the ANT- and BCKD-specific rabbit and DCM sera react with small foci of cardiac myocytes of DCM patients, it is not clear at present whether this reactivity is against these molecules or due to cross reactivity. It is the objective of this proposal to (1) biochemically define the true identity of the reactive cardiac tissue autoantigens that are recognized by T cells and sera from DCM patients; (2) map the T and B cell epitopes of these autoantigens; (3) determine the molecular basis for the immunogenic expression of these autoantigens; (4) determine the feasibility of using the established human cardiac myocyte cell lines as a surrogate for human myocytes to study the expression of the putative autoantigens post experimental infection in vitro with CVB3 or treatment with cardiotoxic drugs such as adriamycin/actinomycin-D, all of which have been previously implicated as the etiological agents in human DCM; and (5) determine the frequency of autoantigen-specific T cells and to correlate this with disease severity and prognosis. It is hoped that these strategies will at least provide further insights into the pathogenesis of human DCM.